Isolation of A2 adenosine receptors from rat brain membranes have been performed. The purification methods included solubilization by digitonin and column chromatographies using Q- Sepharose, hydroxylapatite, Green A-agarose and Mono Q. Approximately 1000-fold purification was achieved at the last stage of purification and the specific binding activity of the final preparation was approximately 100 pmol/mg protein assayed using (3H)N-ethylcarboxamideadenosine (NECA) as a ligand. The apparent molecular weight of the partially purified A2 adenosine receptors was estimated to be approximately 80,000 by HPLC (TSK 4000PW-5000PW) experiments. It was also found that A2 adenosine receptors could be separated from A1 adenosine receptors or other unknown adenosine binding sites by hydroxylapatite chromatography. Mouse mastocytoma P815 cell membranes were demonstrated to have A2 adenosine binding sites in a relatively high concentration. These adenosine binding sites were also solubilized by detergents without any significant changes of ligand binding properties.